Complement is the collective term for a series of blood proteins and is a major effector mechanism of the immune system. Complement activation and its deposition on target structures can lead to direct complement-mediated cell lysis, or can lead indirectly to cell or tissue destruction due to the generation of powerful modulators of inflammation and the recruitment and activation of immune effector cells. Complement activation products that mediate tissue injury are generated at various points in the complement pathway. Inappropriate complement activation on host tissue plays an important role in the pathology of many autoimmune and inflammatory diseases, and is also responsible for many disease states associated with bioincompatibility, e.g. post-cardiopulmonary inflammation and transplant rejection. Complement inhibition represents a potential therapeutic modality for the treatment of such immune-mediated diseases and disease states. Complement inhibitory proteins that systemically inhibit complement have been shown to be effective in various animal models of disease (and in a few clinical trials), but complement inhibitors that target a site of disease and complement activation offer significant potential advantages with regard to safety and efficacy.
In healthy individuals, complement deposition on host cell membranes is prevented by complement inhibitory proteins expressed at the cell surface. These complement inhibitory proteins are also expressed on the surface of tumor cells, often at increased levels, and are considered to be an important contributing factor to the resistance of tumor cells to monoclonal antibody-mediated immunotherapy (monoclonal antibodies that target to tumor cells and activate complement).
The complement system comprises a collection of about 30 proteins and is one of the major effector mechanisms of the immune system. The complement cascade is activated principally via either the classical (usually antibody-dependent) or alternative (usually antibody-independent) pathways. Activation via either pathway leads to the generation of C3 convertase, which is the central enzymatic complex of the cascade. C3 convertase cleaves serum C3 into C3a and C3b, the latter of which binds covalently to the site of activation and leads to the further generation of C3 convertase (amplification loop). The activation product C3b (and also C4b generated only via the classical pathway) and its breakdown products are important opsonins and are involved in promoting cell-mediated lysis of target cells (by phagocytes and NK cells) as well as immune complex transport and solubilization. C3/C4 activation products and their receptors on various cells of the immune system are also important in modulating the cellular immune response. C3 convertases participate in the formation of C5 convertase, a complex that cleaves C5 to yield C5a and C5b. C5a has powerful proinflammatory and chemotactic properties and can recruit and activate immune effector cells. Formation of C5b initiates the terminal complement pathway resulting in the sequential assembly of complement proteins C6, C7, C8 and (C9)n to form the membrane attack complex (MAC or C5b-9). Formation of MAC in a target cell membrane can result in direct cell lysis, but can also cause cell activation and the expression/release of various inflammatory modulators.
There are two broad classes of membrane complement inhibitor; inhibitors of the complement activation pathway (inhibit C3 convertase formation), and inhibitors of the terminal complement pathway (inhibit MAC formation). Membrane inhibitors of complement activation include complement receptor 1 (CR1), decay-accelerating factor (DAF) and membrane cofactor protein (MCP). They all have a protein structure that consists of varying numbers of repeating units of about 60-70 amino acids termed short consensus repeats (SCR) that are a common feature of C3/C4 binding proteins. Rodent homologues of human complement activation inhibitors have been identified. The rodent protein Crry is a widely distributed inhibitor of complement activation that functions similar to both DAF and MCP. Rodents also express DAF and MCP, although Crry appears to be functionally the most important regulator of complement activation in rodents. Although there is no homolog of Crry found in humans, the study of Crry and its use in animal models is clinically relevant.
Control of the terminal complement pathway and MAC formation in host cell membranes occurs principally through the activity of CD59, a widely distributed 20 kD glycoprotein attached to plasma membranes by a glucosylphosphatidylinositol (GPI) anchor. CD59 binds to C8 and C9 in the assembling MAC and prevents membrane insertion.
Various types of complement inhibitory proteins are currently under investigation for therapy of inflammatory disease and disease states associated with bioincompatibility. Two of the best therapeutically characterized inhibitors of human complement are a soluble form of complement receptor 1 (sCR1) and an anti-05 monoclonal antibody. These systemically active inhibitory proteins have shown efficacy in various animal models of disease and more recently in clinical trials (1-5, 6:#1037). Anti-05 mAb inhibits the generation of C5a and the MAC, whereas sCR1 is an inhibitor of complement activation and also inhibits the generation of C3 activation products. Soluble forms of human decay accelerating factor (DAF) and membrane cofactor protein (MCP), membrane inhibitors of complement activation, have also been shown to be protective an animal models of inflammation and bioincompatability (7-11). CD59 is a membrane inhibitor of complement that blocks assembly of the MAC, but does not effect generation of complement opsonins or C3a and C5a. Soluble forms of CD59 have been produced, but its low functional activity in vitro, particularly in the presence of serum, indicates that sCD59 will have little or no therapeutic efficacy (12-15).
Targeting complement inhibitors to sites of complement activation and disease is likely to improve their efficacy. Since complement plays an important role in host defense and immune complex catabolism, targeted complement inhibitors can also reduce potentially serious side effects, particularly with long term complement inhibition. Recently, a modified form of sCR1 decorated with sialyl Lewis x (sLex) was prepared and shown to bind to endothelial cells expressing P and E selectin. sCR1 sLex was shown to be a more potent therapeutic than sCR1 in rodent models of inflammatory disease (16, 17). In in vitro feasibility studies, antibody-DAF (18) and antibody-CD59 (19) fusion proteins were shown to be more effective at protecting targeted cells than untargeted cells from complement. Non-specific membrane targeting of recombinant complement inhibitors has also been achieved by coupling inhibitors to membrane-inserting peptides (20, 21).
C3 activation fragments are abundant complement opsonins found at a site of complement activation, and they serve as ligands for various C3 receptors. One such receptor, complement receptor 2 (CR2), a transmembrane protein, plays an important role in humoral immunity by way of its expression predominantly on mature B cells and follicular dendritic cells (22, 23). CR2 is a member of the C3 binding protein family and consists of 15-16 short consensus repeat (SCR) domains, structural units that are characteristic of these proteins, with the C3 binding site being contained in the two N-terminal SCRs (24, 25). CR2 is not an inhibitor of complement and it does not bind C3b, unlike the inhibitors of complement activation (DAF, MCP, CR1 and Crry). Natural ligands for CR2 are iC3b, C3dg and C3d, cell-bound breakdown fragments of C3b that bind to the two N-terminal SCR domains of CR2 (26, 27). Cleavage of C3 results initially in the generation and deposition of C3b on the activating cell surface. The C3b fragment is involved in the generation of enzymatic complexes that amplify the complement cascade. On a cell surface, C3b is rapidly converted to inactive iC3b, particularly when deposited on a host surface containing regulators of complement activation (ie. most host tissue). Even in absence of membrane bound complement regulators, substantial levels of iC3b are formed. iC3b is subsequently digested to the membrane bound fragments C3dg and then C3d by serum proteases, but this process is relatively slow (28, 29). Thus, the C3 ligands for CR2 are relatively long lived once they are generated and will be present in high concentrations at sites of complement activation.